Carotenoids are the most widespread class of naturally occurring pigments in nature, present without exception in photosynthetic tissue and occurring with no definite pattern in non-photosynthetic tissues such as roots, flower petals, seeds and fruits. They are also found in algae, fungi, yeasts, molds, mushrooms and bacteria. They represent the most extended group of natural pigments that exists in nature. They are used in the industry as food supplements and colorants. They are known for their excellent anti-oxidant properties and as precursors of vitamin A.
Beta-carotene and Lycopene are important carotenoids used widely in Nutraceutical, Pharma, food and animal feed industries as natural antioxidants, functional colour and for food and feed fortification. High purity Carotenoid crystals plays a major role in the various formulations like encapsulation, water soluble powder or emulsion, high concentrate oil dispersion etc.
These high purity carotenoid crystals not only facilitate easier product formulation but also help to deliver high concentrated end products to meet the growing market demands of anti-oxidants and ‘natural, colorants—a number of methods have been proposed and tried—to isolate and purify carotenoids and specifically Beta carotene.
Carotenoids and Beta carotene, in particular can be obtained from natural sources whether vegetal products such as tomato and carrot in which they are very small percentages or starting from cultures of selected algae, fungi etc. in which proportion of these components may increase.
Beta carotene obtained from fermentation broths of certain mucor fungi such as Phycomyces; Blakeslea etc. have certain advantages over the aforementioned natural sources. ie. 1. Elevated concentration of this compound with respect to the quantity of dry biomasses 2. Possible increase in production using ‘enhanced carotenoid producing strains’ obtained by classical mutagenesis/molecular biology techniques; optimisation of fermentation processes, use of “inducers,/‘Inhibitors’.
For example: U.S. Pat. No. 2,959,521 describes enhanced production of beta carotene from fungi Blakeslea trispora in a culture medium containing Lecithin.
U.S. Pat. No. 7,799,540 Describes a process of producing lycopene through the fermentation of selected strains of Blakeslea trispora and extracting lycopene by a process of treating the fermented culture with alcohol and drying the biomass followed by a mechanical milling process to break the cell wall and the disrupted biomass is extracted by an organic solvent and the crystals are recovered by precipitation crystallization by addition of alcohol. The main disadvantage of this process is the mechanical milling method for cell disruption which might cause the loss of Lycopene;
U.S. Pat. No. 7,252,965 Discloses a method of production of Beta-carotene by fermentation of (+) and (−) strains of Blakeslea trispora followed by separation of the wet biomass and treatment with alcohol to dry the biomass for cell rupturing; then the ruptured cell biomass is subjected to solid-liquid extraction with an organic solvent and it is concentrated. Precipitation/crystallization—by adding alcohol thus a pure crystal of purity >95% is obtained. (Similar process is also explained in US application U.S. Pat. Application No. 20040067550) the disadvantage of this process is the mechanical disruption & drying of the biomass which might cause loss of beta-carotene and complexity of using multiple solvents for extraction and precipitation steps.
U.S. Pat. No. 7,015,014 (equivalent to WO 01/55100) teaches a method for the Isolation of Carotenoid crystals by microbial cell disruption to get an oily medium; this oily suspension is treated with water and the PH adjusted by adding alkali and further treated with alcohol or salt to get a filtrate containing beta-carotene crystals. This filtrate is subjected to several washing steps with multiple solvents followed by a separation and dryings step to get purified crystals. This process involves use of larger number of solvents and water media for the purification
International patent application WO 03/038064 A2 describes method of producing lycopene by fermentation of mutated Blakeslea trispora. Lycopene crystals are extracted from the culture broth by means of cell disruption subsequent purification steps using organic solvents such as ethyl acetate, hexane and 1-butanol by adjusting the PH using alkali with optional addition of alcohol or acid to adjust the pH; this is further purified with organic solvent and washed with fresh solvent to give lycopene crystals. This process is highly complex, larger amount of solvents and reagents are used for the purification steps.
U.S. Pat. No. 6,812,001 (equitant to EP 0979302 & WO 01/83437 A1) Describes a method to obtain carotenoid crystals from microbial biomass, the method involves cell wall disruption by homogenization and followed by lipid removal by alcohol treatment. The crystals from the cellular debris is collected by floating in water. According to this method ether salt or oil can be used in the place of water to float crystals, the yield thus obtained is 35% only.
WO 98/50574 Teaches a method of isolating carotenoid crystals from microbial biomass which involves disrupting the microbial cell walls, removing the cellular debris from the resulting carotenoid crystal containing residue. Further solvent added to remove lipid. Then Carotenoids are extracted using ethyl acetate, hexane or oil followed by several purification & washing steps which requires more solvent & water in the process, thus the crystals purity of 93.3% is achieved with an yield of 35% only.
U.S. Pat. No. 5,858,700 Teaches a process for the purification of lycopene crystals by saponifying lycopene containing oleoresin using propylene glycol, alkali and water to recover the crystals
U.S. Pat. No. 5,714,658, Describes a process for the extraction of carotenes from natural sources using a mixture of an acetic acid ester (ethyl acetate or butyl acetate or in combination), and with an edible oil.
U.S. Pat. No. 3,268,606 Describes a process of extracting beta-carotene from fungal biomass by treating the fermented mycelium with alcohol to remove the moisture and extracting the dried mycelium with ethylene chloride or Benzene in several steps to yield a filtrate rich in beta-carotene. This filtrate is further concentrated and subjected to crystallization with acetone and absolute alcohol to recover the high purity beta-carotene crystals. The disadvantage of this process is the complexity in industrial application, usage of toxic solvents like benzene.
U.S. Pat. Application No. 20020025548 (equivalent to WO/1998/003480) discloses a method to extract beta carotene from natural sources by direct extraction with organic solvents, vegetable oils or supercritical fluids, followed by crystallization or precipitation and washing of the crystals with an anti-solvent.
U.S. Pat. Application No. 20060105443 explains a method to isolate lycopene from transformed or naturally derived bacterial cells; the steps in the process comprises: isolating a biomass from a fermented broth; treating the isolated biomass with alcohol; extracting the lycopene from the alcohol treated biomass with methylene chloride; solid mixture is removed and the filtrate is vacuum contracted; the concentrates suspension is washed with acetone to recover lycopene crystals.
U.S. Pat. Application No. 20060234333 (equivalent to WO/2004/063359) discloses a method for producing carotenoids or their precursors using genetically modified organisms of the blakeslea genus. The process involves recovery of biomass from the medium and washing the same with water. Biomass is sterilized and cell disruptions are carried out using steam or microwave radiation followed by extraction using solvents such as dichloromethane or supercritical carbon dioxide or tetrahydrofuran. The filtrate obtained after the extraction is subjected to crystallization step using a carotenoid non soluble solvent.
CN101870668 Discloses a method for preparing beta-carotene from Blakeslea trispora through fermentation & drying the biomass and milling and pulverizing the dry mycelia, followed by solvent extraction using dichloromethane. The major disadvantage of this process is mechanical cell disruption using milling and pulverizing which will lead to loss of beta-carotene.
U.S. Pat. Application No. 20100145116 (equivalent to WO/2008/108674) Describes a process for the production and extraction of Carotenoids without cell disruption by direct solid liquid extraction using a ketone and alcohol (acetone and ethanol or acetone and methanol) followed by a second extraction using a mixture of hydrophobic solvents (hexane and tert-butylmethyl ethe) and finally crystallization. The major disadvantage of the process is using more number of solvents, multiple extraction steps and complicated process.
It is clearly evident that the prior art that the disclosed processes involve: different types & large amount of Organic solvents for the extraction of substantially pure crystals of beta-carotene or lycopene, and the methods are complicated. In most of the cases the purity of the final product is below expectation. The processes for obtaining Beta carotene from fermentation broths described until present generally imply an extraction stage and successive crystallizations and re-crystallizations and even stages of Chromatographic purifications requiring high consumption of solvents. Hence there is a need for a simple, cost effective and industrially viable method for extraction.